Search

Astrocytes are abundant cells in the brain and have vital roles in various brain functions that include biochemical support of endothelial cells, supplying nutrients to the nervous tissue, maintaining the extracellular ion balance, etc. In developing nervous tissue, the differentiation of astrocytes occurs later compared to neurons. It takes more time and more techniques to obtain mature and pure astrocytes in vitro. In this study, a protocol was developed to culture mature and pure astrocytes from human embryonic stem cells (hESCs). To obtain a high quantity and quality of differentiated astrocytes, first, we efficiently generated neural progenitor cells (NPCs) derived from hESCs through the process of embryoid body (EB) formation by adding SB431542 and LDN193189 and neurosphere step. In the astrocyte differentiation stage, the efficiency of astrocyte differentiation was increased using progenitor medium containing EGF and heparin and astrocyte defined medium containing ciliary neurotrophic factor (CNTF). The cell properties were checked with immunocytochemistry and western blot using antibodies for astrocyte-specific marker proteins. From the FACS analysis, we found that the percentage of astrocytes among the cells differentiated from NPCs was over 80%. To validate the functional properties of the astrocytes, we checked IL-6 release from the astrocytes and support of synaptic formation in a co-culture with neurons. Taken altogether, with our protocol, we obtained mature astrocytes within 4 weeks from NPCs and 6 weeks from hESCs.